The present invention relates to methods of screening for drug binding to serum proteins using spectrofluorimetry. Kits useful for performing fluorimetric screening of drug binding to serum proteins are also disclosed.
Potent, pharmacologically active new drug candidates can be effective in vivo only if they are able to achieve and maintain therapeutic concentrations at the site of action. Pharmaceutical properties such as solubility, partition coefficient, permeability, and protein binding contribute to in vivo disposition and, frequently, these properties are important determinants of clinical outcome. The recent successes of combinatorial chemistry in accelerating drug discovery have also increased the interest in rapid, resource-sparing approaches to determining pharmaceutical properties.
The binding of drugs to serum proteins is particularly important, because it affects both the activity of drugs and their disposition (Huang et al., xe2x80x9cEffect of Altered Disopyramide Binding on its Pharmacologic Response in Rabbits,xe2x80x9d Journal of Pharmacology and Experimental Therapeutics, 223:469-71 (1982); Qin et al., xe2x80x9cDecreased Elimination of Drug in the Presence of Alpha-1-acid Glycoprotein is Related to a Reduced Hepatocyte Uptake,xe2x80x9d Journal of Pharmacology and Experimental Therapeutics, 269:1176-81 (1994)). According to the xe2x80x9cfree drugxe2x80x9d hypothesis, only unbound drug exerts pharmacological activity (Recant et al., xe2x80x9cThyroid Function in Nephrosis,xe2x80x9d Journal of Clinical Investigation, 31:789 (1952)) and disposition is often altered by drug binding (Shand et al., xe2x80x9cPerfusion-Limited of Plasma Drug Binding on Hepatic Drug Extraction,xe2x80x9d Life Sciences, 19:125-30 (1976); Jansen, xe2x80x9cInfluence of Plasma Protein Binding Kinetics on Hepatic Clearance Assessed from a xe2x80x9cTubexe2x80x9d Model and a xe2x80x9cWell-stirredxe2x80x9d Model,xe2x80x9d Journal of Pharmacokinetics and Biopharmaceutics, 9:15-26 (1981)). Consequently, it is important to know the affinity of a drug for serum proteins.
A variety of techniques have been proposed for protein binding measurements including dialysis, ultrafiltration (Huang, xe2x80x9cErrors in Estimating the Unbound Fraction of Drugs Due to the Volume Shift in Equilibrium Dialysis,xe2x80x9d Journal of Pharmaceutical Sciences, 72:1368-9 (1983)), circular dichroism (Ascoli et al., xe2x80x9cStereospecific and Competitive Binding of Drugs to Human Serum Albumin: A Difference Circular Dichroism Approach,xe2x80x9d Journal of Pharmaceutical Sciences, 84:737-41 (1995)), and extrinsic fluorescence (Sudlow et al., xe2x80x9cSpectroscopic Techniques in the Study of Protein Binding: The Use of 1-Anilino-8-Naphthalenesulphonate as a Fluorescent Probe for the Study of the Binding of lophenoxic and lopanoic Acids to Human Serum Albumin,xe2x80x9d Molecular Pharmacology, 9:649-57 (1973); Sudlow et al., xe2x80x9cThe Characterization of Two Specific Drug Binding Sites on Human Serum Albumin,xe2x80x9d Molecular Pharmacology, 11:824-32(1975); Epps et al., xe2x80x9cA General, Wide-range Spectrofluorometric Method for Measuring the Site-Specific Affinities of Drugs Toward Human Serum Albumin,xe2x80x9d Analytical Biochemistry, 227:342-50 (1995); Suarez Varela et al., xe2x80x9cSpectrofluorimetric Study of the Binding of 1-Anilnonaphthalene-8-Sulfonate to Bovine Serum Albumin,xe2x80x9d Journal of Pharmaceutical Sciences, 81:843-4 (1992)). Despite the fact that the displacement of extrinsic fluorophores such as warfarin and dansylglycine has been proposed as the basis for a rapid protein binding assay (Epps et al., xe2x80x9cA General, Wide-range Spectrofluorometric Method for Measuring the Site-Specific Affinities of Drugs Toward Human Serum Albumin,xe2x80x9d Analytical Biochemistry, 227:342-50 (1995)) and the fact that such assays are drug nonspecific and rapid, they are indirect because they utilize the interaction between two drugs to produce an extrinsic signal.
The present invention is directed to overcoming these deficiencies in the art.
The present invention relates to a method of screening for drug binding to serum proteins. This method includes preparing at least two solutions, each of the at least two solutions containing a concentration of a serum protein characterized by broad specificity in binding to xenobiotics and a concentration of a candidate drug, wherein the concentration of the candidate drug is different for each of the at least two solutions and, optionally, one of the at least two solutions is a control solution characterized by a candidate drug concentration of zero; exposing each of the at least two solutions to a light source; measuring fluorescent emission by the serum protein or a serum protein-candidate drug complex for each of the at least two solutions upon the exposing; and determining whether a change in fluorescence emission is measured for an increased concentration of the candidate drug, wherein the change in fluorescence emission indicates binding of the candidate drug to the serum protein.
The present invention also relates to a method of screening for drug binding to serum proteins, where a dissociation constant (Kd) for the candidate drug and the serum protein can be calculated based on the measured fluorescence emissions. This method includes preparing at least two solutions, each of the at least two solutions containing a concentration of a serum protein characterized by broad specificity in binding to xenobiotics and a concentration of a candidate drug, wherein the concentration of the candidate drug is different for each of the at least two solutions and, optionally, one of the at least two solutions is a control solution characterized by a candidate drug concentration of zero; exposing each of the at least two solutions to a light source; measuring fluorescent emission by the serum protein or a serum protein-candidate drug complex for each of the at least two solutions upon the exposing; and calculating a dissociation constant (Kd) for the candidate drug and the serum protein based on the measured fluorescence emissions.
Another aspect of the present invention relates to a kit useful for performing a fluorimetric screening of drug binding to serum proteins. The kit includes a plurality of detection cells compatible for use with a fluorimetric device, one or more solutions each having a predetermined concentration of a serum protein characterized by broad specificity in binding to xenobiotics, and instructions for combining a volume of the one or more solutions with a quantity of a drug in the detection cells, exposing the detection cells to the fluorimetric device, and analyzing fluorimetric emission data.
The present invention uses spectrofluorimetry, a technique that has been widely used to study biomolecular interactions and which has many advantages over other techniques such as dialysis and ultrafiltration. The advantages arise primarily because fluorescence data are obtained without separating the bound and unbound species, which reduces the time required for the experiment and eliminates the need for a size-selective membrane. The dialysis and ultrafiltration methods require analysis of free and total drug concentration which can be resource and time consuming. Additionally, these methods cannot be used with drugs that bind extensively to the membrane (MacKichan, xe2x80x9cInfluence of Protein Binding and Use of Unbound (Free) Drug Concentrationsxe2x80x9d in Applied Pharmacokinetics: Principles of Therapeutic Drug Monitoring, pp 5.1-5.48, Evans et al. (eds.), Applied Therapeutics, Vancouver, Wash., (1992), which is hereby incorporated by reference in its entirety); this is often a serious problem with highly hydrophobic drugs. Although the displacement of extrinsic fluorophores such as warfarin and dansylglycine has been proposed as the basis for a rapid protein binding assay (Epps et al., xe2x80x9cA General, Wide-range Spectrofluorometric Method for Measuring the Site-Specific Affinities of Drugs Toward Human Serum Albumin,xe2x80x9d Analytical Biochemistry, 227:342-50 (1995), which is hereby incorporated by reference in its entirety), intrinsic fluorescence offers advantages over extrinsic fluorescence and has the potential to yield better estimates of a dissociation constant (Kd) when, for example, the drug of interest is physically incompatible with the fluorophores or if there are interactions between the binding sites. Physical incompatibility could occur, for example, if the drug of interest caused the fluorophore to precipitate. The present invention also requires only small sample volumes, is amenable to automation, and may be carried out using a multiwell format. Finally, the present invention is not compound specific and, thus, does not require specific drug analysis such as analytical chromatography or mass spectrometry for quantitation.